Hedgehog (Hh) proteins are understood as a family of secreted signal proteins which are responsible for the formation of numerous structures in embryogenesis (J. C. Smith, Cell 76 (1994) 193 196, N. Perrimon, Cell 80 (1995) 517 520, C. Chiang et al., Nature 83 (1996) 407, M. J. Bitgood et al., Curr. Biol. 6 (1996) 296, A. Vortkamp et al., Science 273 (1996) 613, C. J. Lai et al., Development 121 (1995) 2349). During its biosynthesis a 20 kD N-terminal domain and a 25 kD C-terminal domain are obtained after cleavage of the signal sequence and autocatalytic cleavage. In the naturally occurring protein the N-terminal domain is modified with cholesterol at its C-terminus after cleavage of the C-terminal domain (J. A. Porter et al., Science 274 (1996) 255 259). In higher life-forms the Hh family is composed of at least three members namely sonic, indian and desert Hh (sHh, IHh, DHh; M. Fletz et al., Development (Suppl.) (1994) 43 51). Differences in the activity of hedgehog proteins that were produced recombinantly were observed after production in prokaryotes and eukaryotes (M. Hynes et al., Neuron 15 (1995) 35 44 and T. Nakamura et al., Biochem. Biophys. Res. Comm. 237 (1997) 485 469).
Improvements in the specificity of agents used to treat various disease states such as cancer, metabolic, and inflammatory diseases is of considerable interest because of the therapeutic benefits which would be realized if the side effects associated with the administration of these agents could be reduced. Traditionally, dramatic improvements in the treatment of cancer are associated with identification of therapeutic agents acting through novel mechanisms.
Aberrant hedgehog (Hh) pathway signaling has been implicated in human malignancies ranging from semi-malignant tumors of the skin to highly aggressive cancers of the brain, lung, pancreas, breast, prostate and lymphoid lineages (Rubin, L. L. and de Sauvage, F. J. Nat Rev Drug Discov, 2006, 5, 1026-1033). Dysregulation of this pathway contributes to uncontrolled proliferation, invasion, metastasis, evasion of apoptosis and resistance to chemotherapy treatments through regulation of the GLI family of transcription factors (GLI1-3), which reside at the distal end of the pathway (Kasper, M., et. al. Eur J Cancer, 2006, 42, 437-445). The initial evidence that aberrant Hh signaling plays a critical role in cancer initiation and/or progression came from the observations that important regulatory pathway components are mutated in a number of cancers. These include loss-of-function mutations in the 12-transmembrane Hh receptor, patched1 (PTCH1) and activating mutations in the 7-transmembrane “GPCR-like” protein smoothened (SMO) observed in basal cell carcinomas, medulloblastomas and rhabdomyosaracomas. (See Johnson et. al. Science, 272: 1668-1671, 1996; Hahn, et. al. Cell, 85: 841-851, 1996; Unden, et. al. Cancer Res, 56: 4562-4565, 1996; and Chidambaram, et. al. Cancer Res, 56: 4599-4601, 1996).
Where a Sporadic loss-of-function mutation in PTCH1 is observed, these cancers are implicated: basal cell carcinomas (Wolter, M., et. al. Cancer Res, 1997, 57, 2581-2585; Reifenberger, J., et. al. Cancer Res, 1998, 58, 1798-1803; Lam, C. W., et. al. Oncogene, 1999, 18, 833-836; Couve-Privat, S., et. al. Cancer Res, 2002, 62, 7186-7189; Xie, J., et. al. Nature, 1998, 391, 90-92), medulloblastomas (Wolter, M., et. al. Cancer Res, 1997, 57, 2581-2585; Raffel, C., et. al. Cancer Res, 1997, 57, 842-845; Pietsch, T., et. al. Cancer Res, 1997, 57, 2085-2088; Vorechovsky, I., et. al. Oncogene, 1997, 15, 361-366; Couve-Privat, S., et. al. Cancer Res, 2002, 62, 7186-7189; Xie, J., et. al. Nature, 1998, 391, 90-92), breast carcinomas (Xie, J., et. al. Cancer Res, 1997, 57, 2369-2372), meningiomas (id.), and rhabdomyosarcoma (Calzada-Wack, J., et. al. Hum Mutat, 2002, 20, 233-234).
In addition, the activity of the Hh pathway has been shown to be critical for the growth and metastasis of a number of tumors that do not contain mutations within any of the defined pathway components including those of the pancreas (Bennan, D. et. al. Nature, 425: 846-851, 2003; Thayer, S. P., et. al. Nature 2003, 425, 851-856; Pasca di Magliano, M., Genes Dev, 20: 3161-3173, 2006; and Gao, J., et. al. Gene Ther, 13: 1587-1594, 2006), prostate (Karhadkar, S. S., et. al. Nature, 431: 707-712, 2004; Sanchez, P., et. al. Proc Natl Acad Sci USA, 101: 12561-12566, 2004; Sheng, T., et. al. Mol Cancer, 3: 29, 2004; and Fan, L., et. al. Endocrinology, 145: 3961-3970, 2004), digestive tract (Berman, D. et. al. Nature, 425: 846-851, 2003; Thayer, S. P., et. al. Nature 2003, 425, 851-856; Fukaya, M., et. al. Gastroenterology, 131: 14-29, 2006; Ohta, M., et. al. Cancer Res, 65: 10822-10829, 2005), and small cell lung cancers (Watkins, D. N., et. al. Nature, 422: 313-317, 2003), and non-small cell lung cancers (Yuan, Z., et. al. Oncogene, 26: 1046-1055, 2007).
The Hh pathway components are implicated in esophageal cancer (Ma, X., et. al. Int J Cancer, 118: 139-148, 2006; Berman, D. et. al. Nature, 425: 846-851, 2003) and are highly expressed in the vast majority (87%, n=43) of chemotherapy-resistant esophageal cancer specimens (Sims-Mourtada, J. et. al. Clin Cancer Res, 12: 6565-6572, 2006). Other cancers where the Hh pathway are involved include biliary tract cancers (Berman, D. et. al. Nature, 425: 846-851, 2003), melanoma (Stecca, B., et. al. Proc Natl Acad Sci USA, 104: 5895-5900, 2007), and stomach cancer (Berman, D. et. al. Nature, 425: 846-851, 2003; Ma, X., et. al. Carcinogenesis, 26: 1698-1705, 2005). Tumors that contain highly proliferative “tumor stem cells” and which represent areas of therapy include glial cell cancers (Clement, V., et. al. Curr Biol, 17: 165-172, 2007), prostate cancers (Li, C., Heidt, et. al. Cancer Res, 67: 1030-1037, 2007), breast cancers (Liu, S., et. al. Cancer Res, 66: 6063-6071, 2006), multiple myelomas (Peacock, C. D., et. al. PNAS, 104: 4048-4053, 2007), and colon cancers (Ricci-Vitiani, L., et. al. Nature, 445: 111-115, 2007).
Finally, the Hh pathway is an essential regulator of “cancer stem cells (CSC)”, which are discrete tumor cell populations that display highly enhanced survival, self-renewal, and tumorigenicity properties (Beachy, P. A., et. al. Nature, 432: 324-331, 2004). Activation of the Hh pathway has been shown to be critical for CSCs of the breast (Liu, S., et. al. Cancer Res, 66: 6063-6071, 2006), central nervous system (Clement, V., Curr Biol, 17: 165-172, 2007) as well as in hematological malignancies (Peacock, C. D., PNAS, 104: 4048-4053, 2007). These cells, in some experimental contexts, have shown to confer resistance to currently used chemotherapy (Bao, S., et. al. Nature, 444: 756-760, 2006; Dean, M., et. al. Nat Rev Cancer, 5: 275-284, 2005). Therefore, a Hh pathway inhibitor may have broad clinical utility treating of a wide range of chemotherapy-resistant malignancies.
In view of the important role of the Hedgehog pathway in biological processes and disease states, modulators of this pathway are desirable.